Isolated human drug-metabolizing proteins, nucleic acid molecules encoding human drug-metabolizing proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the proteins of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the proteins of the present invention, and methods of identifying modulators of the proteins of the present invention.

FIELD OF THE INVENTION

The present invention is in the field of proteins that are related tothe sulfotransferase drug-metabolizing enzyme subfamily, recombinant DNAmolecules and protein production. The present invention specificallyprovides novel drug-metabolizing peptides and proteins and nucleic acidmolecules encoding such protein molecules, for use in labeling reagentswith tagged sulfer and for the development of human therapeutics andhuman therapeutic development.

BACKGROUND OF THE INVENTION

Drug-Metabolizing Proteins

Induction of drug-metabolizing enzymes (“DMEs”) is a common biologicalresponse to xenobiotics, the mechanisms and consequences of which areimportant in academic, industrial, and regulatory areas of pharmacologyand toxicology.

For most drugs, drug-metabolizing enzymes determine how long and howmuch of a drug remains in the body. Thus, developers of drugs recognizethe importance of characterizing a drug candidate's interaction withthese enzymes. For example, polymorphisms of the drug-metabolizingenzyme CYP2D6, a member of the cytochrome p450 (“CYP”) superfamily,yield phenotypes of slow or ultra-rapid metabolizers of a wide spectrumof drugs including antidepressants, antipsychotics, beta-blockers, andantiarrhythmics. Such abnormal rates of drug metabolism can lead to drugineffectiveness or to systemic accumulation and toxicity.

For pharmaceutical scientists developing a candidate drug, it isimportant know as early as possible in the design phase which enzymesmetabolize the drug candidate and the speed with which they do it.Historically, the enzymes on a drug's metabolic pathway were determinedthrough metabolism studies in animals, but this approach has now beenlargely supplanted by the use of human tissues or cloneddrug-metabolizing enzymes to provide insights into the specific role ofindividual forms of these enzymes. Using these tools, the qualitativeand quantitative fate of a drug candidate can be predicted prior to itsfirst administration to humans. As a consequence, the selection andoptimization of desirable characteristics of metabolism are possibleearly in the development process, thus avoiding unanticipated toxicityproblems and associated costs subsequent to the drug's clinicalinvestigation. Moreover, the effect of one drug on another's dispositioncan be inferred.

Known drug-metabolizing enzymes include the cytochrome p450 (“CYP”)superfamily, N-acetyl transferases (“NAT”), UDP-glucuronosyltransferases (“UGT”), methyl transferases, alcohol dehydrogenase(“ADH”), aldehyde dehydrogenase (“ALDH”), dihydropyrimidinedehydrogenase (“DPD”), NADPH:quinone oxidoreductase (“NQO” or “DTdiaphorase”), catechol O-methyltransferase (“COMT”), glutathioneS-transferase (“GST”), histamine methyltransferase (“HMT”),sulfotransferases (“ST”), thiopurine methyltransferase (“TPMT”), andepoxide hydroxylase. Drug-metabolizing enzymes are generally classifiedinto two phases according to their metabolic function. Phase I enzymescatalyze modification of functional groups, and phase II enzymescatalyze conjugation with endogenous substituents. These classificationsshould not be construed as exclusive nor exhaustive, as other mechanismsof drug metabolism have been discovered. For example, the use of activetransport mechanisms been characterized as part of the process ofdetoxification.

Phase I reactions include catabolic processes such as deamination ofaminases, hydrolysis of esters and amides, conjugation reactions with,for example, glycine or sulfate, oxidation by the cytochrome p450oxidation/reduction enzyme system and degradation in the fatty acidpathway. Hydrolysis reactions occur mainly in the liver and plasma by avariety of non-specific hydrolases and esterases. Both deaminases andamidases, also localized in the liver and serum, carry out a large partof the catabolic process. Reduction reactions occur mainlyintracellularly in the endoplasmic reticulum.

Phase II enzymes detoxify toxic substances by catalyzing theirconjugation with water-soluble substances, thus increasing toxins'solubility in water and increasing their rate of excretion.Additionally, conjugation reduces the toxins' biological reactivity.Examples of phase II enzymes include glutathione S-transferases andUDP-glucuronosyl transferases, which catalyze conjugation to glutathioneand glucuronic acid, respectively. Transferases perform conjugationreactions mainly in the kidneys and liver.

The liver is the primary site of elimination of most drugs, includingpsychoactive drugs, and contains a plurality of both phase I and phaseII enzymes that oxidize or conjugate drugs, respectively.

Physicians currently prescribe drugs and their dosages based on apopulation average and fail to take genetic variability into account.The variability between individuals in drug metabolism is usually due toboth genetic and environmental factors, in particular, how thedrug-metabolizing enzymes are controlled. With certain enzymes, thegenetic component predominates and variability is associated withvariants of the normal, wild-type enzyme.

Most drug-metabolizing enzymes exhibit clinically relevant geneticpolymorphisms. Essentially all of the major human enzymes responsiblefor modification of functional groups or conjugation with endogenoussubsituents exhibit common polymorphisms at the genomic level. Forexample, polymorphisms expressing a non-functioning variant enzymeresults in a sub-group of patients in the population who are more proneto the concentration-dependent effects of a drug. This sub-group ofpatients may show toxic side effects to a dose of drug that is otherwisewithout side effects in the general population. Recent development ingenotyping allows identification of affected individuals. As a result,their atypical metabolism and likely response to a drug metabolized bythe affected enzyme can be understood and predicted, thus permitting thephysician to adjust the dose of drug they receive to achieve improvedtherapy.

A similar approach is also becoming important in identifying riskfactors associated with the development of various cancers. This isbecause the enzymes involved in drug metabolism are also responsible forthe activation and detoxification of chemical carcinogens. Specifically,the development of neoplasia is regulated by a balance between phase Ienzymes, which activate carcinogens, and phase II enzymes, whichdetoxify them. Accordingly, an individual's susceptibility to canceroften involves the balance between these two processes, which is, inpart, genetically determined and can be screened by suitable genotypingtests. Higher induction of phase I enzymes compared to phase II enzymesresults in the generation of large amounts of electrophiles and reactiveoxygen species and may cause DNA and membrane damage and other adverseeffects leading to neoplasia. Conversely, higher levels of phase IIenzyme expression can protect cells from various chemical compounds.

Abnormal activity of drug-metabolizing enzymes has been implicated in arange of human diseases, including cancer, Parkinson's disease, myetonicdystrophy, and developmental defects.

Cytochrome p450

An example of a phase I drug-metabolizing enzyme is the cytochrome p450(“CYP”) superfamily, the members of which comprise the majordrug-metabolizing enzymes expressed in the liver. The CYP superfamilycomprises heme proteins which catalyze the oxidation and dehydrogenationof a number of endogenous and exogenous lipophilic compounds. The CYPsuperfamily has immense diversity in its functions, with hundreds ofisoforms in many species catalyzing many types of chemical reactions.The CYP superfamily comprises at least 30 related enzymes, which aredivided into different families according to their amino acid homology.Examples of CYP families include CYP families 1, 2, 3 and 4, whichcomprise endoplasmic reticulum proteins responsible for the metabolismof drugs and other xenobiotics. Approximately 10-15 individual geneproducts within these four families metabolize thousands of structurallydiverse compounds. It is estimated that collectively the enzymes in theCYP superfamily participate in the metabolism of greater than 80% of allavailable drugs used in humans. For example, the CYP 1A subfamilycomprises CYP 1A2, which metabolizes several widely used drugs,including acetaminophen, amitriptyline, caffeine, clozapine,haloperidol, imipramine, olanzapine, ondansetron, phenacetin,propafenone, propranolol, tacrine, theophylline, verapamil. In addition,CYP enzymes play additional roles in the metabolism of some endogenoussubstrates including prostaglandins and steroids.

Some CYP enzymes exist in a polymorphic form, meaning that a smallpercentage of the population possesses mutant genes that alter theactivity of the enzyme, usually by diminishing or abolishing activity.For example, a genetic polymorphism has been well characterized with theCYP 2C19 and CYP 2D6 genes. Substrates of CYP 2C19 include clomipramine,diazepam, imipramine, mephenyloin, moclobemide, omeprazole, phenyloin,propranolol, and tolbutamide. Substrates of CYP 2D6 include alprenolol,amitriptyline, chlorpheniramine, clomipramine, codeine, desipramine,dextromethorphan, encainide, fluoxetine, haloperidol, imipramine,indoramin, metoprolol, nortriptyline, ondansetron, oxycodone,paroxetine, propranolol, and propafenone. Polymorphic variants of thesegenes metabolize these substrates at different rates, which can effect apatient's effective therapeutic dosage.

While the substrate specificity of CYPs must be very broad toaccommodate the metabolism of all of these compounds, each individualCYP gene product has a narrower substrate specificity defined by itsbinding and catalytic sites. Drug metabolism can thereby be regulated bychanges in the amount or activity of specific CYP gene products. Methodsof CYP regulation include genetic differences in the expression of CYPgene products (i.e., genetic polymorphisms), inhibition of CYPmetabolism by other xenobiotics that also bind to the CYP, and inductionof certain CYPs by the drug itself or other xenobiotics. Inhibition andinduction of CYPs is one of the most common mechanisms of adverse druginteractions. For example, the CYP3A subfamily is involved in clinicallysignificant drug interactions involving nonsedating antihistamines andcisapride that may result in cardiac dysrhythmias. In another example,CYP3A4 and CYP1A2 enzymes are involved in drug interactions involvingtheophylline. In yet another example, CYP2D6 is responsible for themetabolism of many psychotherapeutic agents. Additionallly, CYP enzymesmetabolize the protease inhibitors used to treat patients infected withthe human immunodeficiency virus. By understanding the unique functionsand characteristics of these enzymes, physicians may better anticipateand manage drug interactions and may predict or explain an individual'sresponse to a particular therapeutic regimen.

Examples of reactions catalyzed by the CYP superfamily includeperoxidative reactions utilizing peroxides as oxygen donors inhydroxylation reactions, as substrates for reductive beta-scission, andas peroxyhemiacetal intermediates in the cleavage of aldehydes toformate and alkenes. Lipid hydroperoxides undergo reductivebeta-cleavage to give hydrocarbons and aldehydic acids. One of theseproducts, trans4-hydroxynonenal, inactivates CYP, particularlyalcohol-inducible 2E1, in what may be a negative regulatory process.Although a CYP iron-oxene species is believed to be the oxygen donor inmost hydroxylation reactions, an iron-peroxy species is apparentlyinvolved in the deformylation of many aldehydes with desaturation of theremaining structure, as in aromatization reactions.

Examples of drugs with oxidative metabolism associated with CYP enzymesinclude acetaminophen, alfentanil, alprazolam, alprenolol, amiodarone,amitriptyline, astemizole, buspirone caffeine, carbamazepine,chlorpheniramine, cisapride, clomipramine, clomipramine, clozapine,codeine, colchicine, cortisol, cyclophosphamide, cyclosporine, dapsone,desipramine, dextromethorphan, diazepam, diclofenac, diltiazem,encainide, erythromycin, estradiol, felodipine, fluoxetine, fluvastatin,haloperidol, ibuprofen, imipramine, indinavir, indomethacin, indoramin,irbesartan, lidocaine, losartan, macrolide antibiotics, mephenyloin,methadone, metoprolol, mexilitene, midazolam, moclobemide, naproxen,nefazodone, nicardipine, nifedipine, nitrendipine, nortriptyline,olanzapine, omeprazole, ondansetron, oxycodone, paclitaxel, paroxetine,phenacetin, phenyloin, piroxicam, progesterone, propafenone,propranolol, quinidine, ritonavir, saquinavir, sertraline, sildenafil,S-warfarin, tacrine, tamoxifen, tenoxicam, terfenadine, testosterone,theophylline, timolol, tolbutamide, triazolam, verapamil, andvinblastine.

Abnormal activity of phase I enzymes has been implicated in a range ofhuman diseases. For example, enhanced CYP2D6 activity has been relatedto malignancies of the bladder, liver, pharynx, stomach and lungs,whereas decreased CYP2D activity has been linked to an increased risk ofParkinson's disease. Other syndromes and developmental defectsassociated with deficiencies in the CYP superfamily includecerebrotendinous xanthomatosis, adrenal hyperplasia, gynecomastia, andmyetonic dystrophy.

The CYP superfamily are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of the CYPsuperfamily.

UDP-Glucuronosyltransferases

Potential drug interactions involving phase II metabolism areincreasingly being recognized. An important group of phase II enzymesinvolved in drug metabolism are the glucuronosyltransferases, especiallythe UDP-glucuronyltransferase (“UGT”) superfamily. Members of the UGTsuperfamily catalyze the enzymatic addition of UDP glucuronic acid as asugar donor to fat-soluble chemicals, a process which increases theirsolubility in water and increases their rate of excretion. In mammals,glucuronic acid is the main sugar that is used to prevent theaccumulation of waste products of metabolism and fat-soluble chemicalsfrom the environment to toxic levels in the body. Both inducers andinhibitors of glucuronosyltransferases are known and have the potentialto affect the plasma concentration and actions of important drugs,including psychotropic drugs.

The UGT superfamily comprises several families of enzymes in severalspecies defined with a nomenclature similar to that used to definemembers of the CYP superfamily. In animals, yeast, plants and bacteriathere are at least 110 distinct known members of the UGT superfamily. Asmany as 33 families have been defined, with three families identified inhumans. Different UGT families are defined as having <45% amino acidsequence homology; within subfamilies there is approximately 60%homology. The members of the UGT superfamily are part of a furthersuperfamily of UDP glycosyltransferases found in animals, plants andbacteria.

The role of phase II enzymes, and of UGT enzymes in particular, is beingincreasingly recognized as important in psychopharmacology. UGT enzymesconjugate many important psychotropic drugs and are an important sourceof variability in drug response and drug interactions. For example, thebenzodiazepines lorazepam, oxazepam, and temazepam undergo phase IIreactions exclusively before being excreted into the urine.

Phase II enzymes metabolize and detoxify hazardous substances, such ascarcinogens. The expression of genes encoding phase II enzymes is knownto be up-regulated by hundreds of agents. For example, oltipraz is knownto up-regulate phase II enzyme expression. Studies have demonstratedprotection from the cancer-causing effects of carcinogens when selectedphase II enzyme inducers are administered prior to the carcinogens. Thepotential use of phase II enzyme inducers in humans for prevention ofcancers related to exposure to carcinogens has prompted studies aimed atunderstanding their molecular effects. Current biochemical and molecularbiological research methodologies can be used to identify andcharacterize selective phase II enzyme inducers and their targets.Identification of genes responding to cancer chemopreventive agents willfacilitate studies of their basic mechanism and provide insights aboutthe relationship between gene regulation, enzyme polymorphism, andcarcinogen detoxification.

Examples of drugs with conjugative metabolism associated with UGTenzymes include amitriptyline, buprenorphine, chlorpromazine, clozapine,codeine, cyproheptadine, dihydrocodeine, doxepin, imipramine,lamotrigine, lorazepam, morphine, nalorphine, naltrexone, temazepam, andvalproate.

Abnormal activity of phase II enzymes has been implicated in a range ofhuman diseases. For example, Gilbert syndrome is an autosomal dominantdisorder caused by mutation in the UGT1 gene, and mutations in theUGT1A1 enzyme have been demonstrated to be responsible forCrigler-Najjar syndrome.

The UGT superfamily are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of the UGTsuperfamily.

Sulfotransferases

Sulfation and sulfate conjugate hydrolysis play an important role inmetabolism, and are catalysed by members of the sulfotransferase andsulfatase enzyme super-families. Sulfotransferases are enzymes thattransfer sulfate groups to acceptor molecules. These enzymes areinvolved in the posttranslational sulfation of proteins and the sulfateconjugation of exogenous chemicals and bile acids. Cytosolicsulfotransferases in mammalian cells catalyze the transfer of asulfonate group from 3′-phosphoadenosine-5′-phosphosulfate, the activesulfate, to the hydroxyl groups or amine groups of substrate compounds.Traditionally, they are viewed as detoxifying or Phase IIdrug-metabolizing enzymes that facilitate the removal of drugs andxenobiotic compounds. Increasingly, however, these enzymes have beenshown to be involved in the sulfation of endogenous compounds, such asthyroid and steroid hormones, catecholamines, and bile acids, to fulfillfundamental biochemical/physiological needs. The balance ofsulfoconjugation (by sulfotransferases) and deconjugation (bysulfotases) may have physiological implications; in addition tocatecholamine release, it may determine the availability of freecatecholamines during diurnal rhythms and stress or modify their renalexcretion. Circumstantial evidence, including a close homology withinthe aryl sulfatases and steroid sulfatase gene, the first implicated incatecholamine metabolism, the second in steroid metabolism, suggests agenetic defect of sulfatases in essential hypertension. A similar, butsecondary, sulfatase defect may affect catecholamine metabolism andaction in chronic renal failure.

In general, sulfation is a deactivating, detoxication pathway, but forsome chemicals the sulfate conjugates are much more reactive than theparent compound (Coughtrie M W, et al., 1998. Biology and function ofthe reversible sulfation pathway catalysed by human sulfotransferasesand sulfatases. Chem Biol Interact. 20;109(1-3):3-27). For exampleN-hydroxyarylamine sulfotransferase (HAST-I) detoxifies phenols butactivates N-hydroxylarylamines into a mutagenic form,N-hydroxy-2-acetylaminofluorene (Nagata, K., et al., 1993. Isolation andexpression of a cDNA encoding a male-specific rat sulfotransferase thatcatalyzes activation of N-hydroxy-2-acetylaminofluorene. J. Biol. Chem.268 (33), 24720-24725 and Gong D W, et al., 1991. Purification ofhepatic N-hydroxyarylamine sulfotransferases and their regulation bygrowth hormone and thyroid hormone in rats. J Biochem (Tokyo) 1991August; 110(2):226-31.).

The discovery of proteins that are related to and are potentially newsulfotransferase satisfies a need in the art by providing newcompositions which are useful towards the design of stable and moreactive drugs, and in the prevention, diagnosis, and treatment of cancer,hypertension, renal diseases, and neurodegenerative diseases.

In addition to such use in the study of drug metabloism,sulfotransferases are also useful in labeling agents with tagged sulfur.This provides another use of the proteins of the present invention.

Further, since the genes of the present invention are mapped tochromosomal position and SNP variation is present, the nucleic acidmolecules of the present invention can be used in linkage studies andgenetic mapping uses.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of aminoacid sequences of proteins that are related to the sulfotransferasedrug-metabolizing enzyme subfamily, as well as allelic variants andother mammalian orthologs thereof. These unique protein sequences, andnucleic acid sequences that encode these proteins, can be used to labelproteins, as models for the development of human therapeutic targets,aid in the identification of therapeutic proteins, and serve as targetsfor the development of human therapeutic agents that modulate protein ofthe present invention activity in cells and tissues that express theprotein of the present invention. Specicially, experimental dataprovided in FIG. 1 shows that the sulfotransferase of the presentinvention is expressed at least in the kidney and small intestine.Furhter, the nucleic acid moelcues herein discribed can be used to labelagents with tagge sulfur.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of two alternative transcriptsequences (SEQ ID NOS:1 and 2) and a cDNA sequence (SEQ ID NO:3) thatencodes the protein of the present invention. In addition, structure andfunctional information is provided, such as ATG start, stop and tissuedistribution, that allows one to readily determine specific uses ofinventions based on this molecular sequence. Experimental data providedin FIG. 1 shows that the sulfotransferase of the present invention isexpressed at least in the kidney and small intestine.

FIG. 2 provides the amino acid sequences of the proteins of the presentinvention. (SEQ ID NOS:4-6) In addition, structural and functionalinformation such as protein family, function, important domains andmodification sites is provided, allowing one to readily determinespecific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding theprotein of the present invention. (SEQ ID NO:7) In addition structuraland functional information, such as intron/exon structure, promoterlocation, etc., is provided allowing one to readily determine specificuses of inventions based on this molecular sequence. For example, asillustrated in FIG. 3, identified SNP variations include a1008c, a6094g,t7898c, a10018c, a10791t, a12451t, t14674c, g14860c, a20133g, a1025c,t12602c, g12829t, a1442t.

DETAILED DESCRIPTION OF THE INVENTION

General Description

The present invention is based on the gene identification that occurredduring the sequencing of the human genome. During the sequencing andassembly of the human genome, analysis of the sequence informationrevealed previously unidentified fragments of the human genome thatencode peptides that share structural and/or sequence homology toprotein/peptide/domains identified and characterized within the art asbeing a drug-metabolizing enzyme protein or part of a drug-metabolizingenzyme protein and are specifically related to the sulfotransferasedrug-metabolizing enzyme subfamily. Utilizing these sequences,additional genomic sequences were assembled and transcript and/or cDNAsequences were isolated and characterized. Based on this analysis, thepresent invention provides amino acid sequences of proteins that arerelated to the sulfotransferase drug-metabolizing enzyme subfamily,nucleic acid sequences in the form of transcript sequences, cDNAsequences and/or genomic sequences that encode these proteins, nucleicacid variation (allelic information), tissue distribution of expression,and information about the closest art known protein/peptide/domain thathas structural or sequence homology to the protein of the presentinvention.

In addition to being previously unknown, the proteins that are providedin the present invention are selected based on their ability to be usedfor the development of commercially important products and services.Specifically, the present proteins are selected based on homology and/orstructural relatedness to known drug-metabolizing enzyme proteins of thesulfotransferase drug-metabolizing enzyme subfamily and the expressionpattern observed. Specifically, experimental data provided in FIG. 1shows that the sulfotransferase of the present invention is expressed atleast in the kidney and small intestine. The art has clearly establishedthe commercial importance of members of this family of proteins andproteins that have expression patterns similar to that of the presentgene for industrial uses, such as labeling agents with tagged sulfur,genetic mapping uses, use of the SNP and chromosomal position forlinkage studies, genetic expression uses, use of the control region todrive expression in the kidneys, etc. Some of the more specific featuresof the proteins of the present invention, and the uses thereof, aredescribed herein, particularly in the Background of the Invention and inthe annotation provided in the Figures, and/or are known within the artfor the known sulfotransferase subfamily of drug-metabolizing enzymeproteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being related to thesulfotransferase drug-metabolizing enzyme subfamily (protein sequencesare provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1and genomic sequences are provided in FIG. 3). The peptide sequencesprovided in FIG. 2, as well as the obvious variants described herein,particularly allelic variants as identified herein and using theinformation in FIG. 3, will be referred herein as the proteins of thepresent invention, or proteins of the present invention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequences of the proteins disclosed in the FIG. 2, (encoded by thenucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The proteins of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thepeptide having less than about 30% (by dry weight) chemical precursorsor other chemicals, less than about 20% chemical precursors or otherchemicals, less than about 10% chemical precursors or other chemicals,or less than about 5% chemical precursors or other chemicals.

The isolated protein can be purified from cells that naturally expressit, purified from cells that have been altered to express it(recombinant), or synthesized using known protein synthesis methods.Experimental data provided in FIG. 1 shows that the sulfotransferase ofthe present invention is expressed at least in the kidney and smallintestine. For example, a nucleic acid molecule encoding the proteins ofthe present invention is cloned into an expression vector, theexpression vector introduced into a host cell and the protein expressedin the host cell. The protein can then be isolated from the cells by anappropriate purification scheme using standard protein purificationtechniques. Many of these techniques are described in detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 2 (SEQ ID NOS:4-6), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NOS:1 and 2, alternative transcript sequences, and SEQ IDNO:3, cDNA sequences) and the genomic sequences provided in FIG. 3 (SEQID NO:7). The amino acid sequence of such a protein is provided in FIG.2. A protein consists of an amino acid sequence when the amino acidsequence is the final amino acid sequence of the protein. Such a proteincan furthe comprise the variants provided in FIG. 3.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 2 (SEQ ID NOS:4-6), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NOS:1 and 2, alternative transcript sequences,and SEQ ID NO:3, cDNA sequences) and the genomic sequences provided inFIG. 3 (SEQ ID NO:7). A protein consists essentially of an amino acidsequence when such an amino acid sequence is present with only a fewadditional amino acid residues, for example from about 1 to about 100 orso additional residues, typically from 1 to about 20 additional residuesin the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 2 (SEQ ID NOS:4-6), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NOS:1 and 2, alternative transcript sequences, and SEQ IDNO:3, cDNA sequences) and the genomic sequences provided in FIG. 3 (SEQID NO:7). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the proteins of the present invention are the naturallyoccurring mature proteins. A brief description of how various types ofthese proteins can be madelisolated is provided below.

The proteins of the present invention can be attached to heterologoussequences to form chimeric or fusion proteins. Such chimeric and fusionproteins comprise one of the proteins of the present inventionoperatively linked to a heterologous protein having an amino acidsequence not substantially homologous to the proteins of the presentinvention. “Operatively linked” indicates that the proteins of thepresent invention and the heterologous protein are fused in-frame. Theheterologous protein can be fused to the N-terminus or C-terminus of theproteins of the present invention.

In some uses, the fusion protein does not affect the activity of theproteins of the present invention per se. For example, the fusionprotein can include, but is not limited to, enzymatic fusion proteins,for example beta-galactosidase fusions, yeast two-hybrid GAL fusions,poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusionproteins, particularly poly-His fusions, can facilitate the purificationof recombinant proteins of the present invention. In certain host cells(e.g., mammalian host cells), expression and/or secretion of a proteincan be increased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A protein of the present invention-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the proteins of the presentinvention.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the proteins of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the proteins of the present invention. Thedegree of homology/identity present will be based primarily on whetherthe peptide is a functional variant or non-functional variant, theamount of divergence present in the paralog family and the evolutionarydistance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, andsplice variants proteins that comprise one of the proteins of thepresent invention can readily be identified as having complete sequenceidentity to at least a portion of one of the proteins of the presentinvention as well as being encoded by the same genetic locus as theproteins of the present invention provided herein. FIG. 3, the mapposition was determined to be on chromosome 2 near marker SHCG-1639 (LODscore 10.01) using radiation hybrid mapping.

Allelic variants of a protein of the present invention can readily beidentified as being a human protein having a high degree (significant)of sequence homology/identity to at least a portion of the proteins ofthe present invention as well as being encoded by the same genetic locusas the proteins of the present invention provided herein. Genetic locuscan readily be determined based on the genomic information provided inFIG. 3, such as the genomic sequence mapped to the reference human. Asindicated by the data presented in FIG. 3, the map position wasdetermined to be on chromosome 2 near marker SHGC-1639 (LOD score 10.01)using radiation hybrid mapping. As used herein, two proteins (or aregion of the proteins) have significant homology when the amino acidsequences are typically at least about 70-80%, 80-90%, and moretypically at least about 90-95% or more homologous. A significantlyhomologous amino acid sequence, according to the present invention, willbe encoded by a nucleic acid sequence that will hybridize to a proteinof the present invention encoding nucleic acid molecule under stringentconditions as more fully described below.

FIG. 3 provides SNP information that was identified from chromosomalsequence obtained from a number of individuals as well as SNPs foundduring the cDNA cloning process. These include, a6094g, t7898c, a10018c,a10791t, a12451t, t14674c, g14860c, a20133g, a1025c, t12602c, g12829t,a1442t.

Paralogs of a protein of the present invention can readily be identifiedas having some degree of significant sequence homology/identity to atleast a portion of the proteins of the present invention, as beingencoded by a gene from humans, and as having similar activity orfunction. Two proteins will typically be considered paralogs when theamino acid sequences are typically at least about 60% or greater, andmore typically at least about 70% or greater homology through a givenregion or domain. Such paralogs will be encoded by a nucleic acidsequence that will hybridize to a protein of the present inventionencoding nucleic acid molecule under moderate to stringent conditions asmore fully described below.

Orthologs of a protein of the present invention can readily beidentified as having some degree of significant sequencehomology/identity to at least a portion of the proteins of the presentinvention as well as being encoded by a gene from another organism.Preferred orthologs will be isolated from mammals, preferably primates,for the development of human therapeutic targets and agents. Suchorthologs will be encoded by a nucleic acid sequence that will hybridizeto a protein of the present invention encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

Non-naturally occurring variants of the proteins of the presentinvention can readily be generated using recombinant techniques. Suchvariants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the proteins of the presentinvention. For example, one class of substitutions are conserved aminoacid substitution. Such substitutions are those that substitute a givenamino acid in a protein of the present invention by another amino acidof like characteristics. Typically seen as conservative substitutionsare the replacements, one for another, among the aliphatic amino acidsAla, Val, Leu, and He; interchange of the hydroxyl residues Ser and Thr;exchange of the acidic residues Asp and Glu; substitution between theamide residues Asn and Gln; exchange of the basic residues Lys and Arg;and replacements among the aromatic residues Phe and Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent are found in Bowie et al., Science 247:1306-1310 (1990).

Variant proteins can be fully functional or can lack function in one ormore activities, e.g. ability to bind substrate, ability tophosphorylate substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variation orvariation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)),particularly using the results provided in FIG. 2. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as drug-metabolizing enzyme activity or in assays such as an invitro proliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

The present invention further provides fragments of the proteins of thepresent invention, in addition to proteins and peptides that compriseand consist of such fragments, particularly those comprising theresidues identified in FIG. 2. The fragments to which the inventionpertains, however, are not to be construed as encompassing fragmentsthat may be disclosed publicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or morecontiguous amino acid residues from a protein of the present invention.Such fragments can be chosen based on the ability to retain one or moreof the biological activities of the proteins of the present invention orcould be chosen for the ability to perform a function, e.g. bind asubstrate or act as an immunogen. Particularly important fragments arebiologically active fragments, peptides that are, for example, about 8or more amino acids in length. Such fragments will typically comprise adomain or motif of the proteins of the present invention, e.g., activesite, a transmembrane domain or a substrate-binding domain. Further,possible fragments include, but are not limited to, domain or motifcontaining fragments, soluble peptide fragments, and fragmentscontaining immunogenic structures. Predicted domains and functionalsites are readily identifiable by computer programs well known andreadily available to those of skill in the art (e.g., PROSITE analysis).The results of one such analysis are provided in FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in proteins are describedin basic texts, detailed monographs, and the research literature, andthey are well known to those of skill in the art (some of these featuresare identified in FIG. 2).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W.H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enymol. 182:626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

Accordingly, the proteins of the present invention also encompassderivatives or analogs in which a substituted amino acid residue is notone encoded by the genetic code, in which a substituent group isincluded, in which the mature protein of the present invention is fusedwith another compound, such as a compound to increase the half-life ofthe protein of the present invention (for example, polyethylene glycol),or in which the additional amino acids are fused to the mature proteinof the present invention, such as a leader or secretory sequence or asequence for purification of the mature protein of the present inventionor a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial andspecific assays related to the functional information provided in theFigures; to label agents with tagged sulfur, to raise antibodies or toelicit another immune response; as a reagent (including the labeledreagent) in assays designed to quantitatively determine levels of theprotein (or its binding partner or ligand) in biological fluids; tometabolize compounds for toxicity studies; and as markers for tissues inwhich the corresponding protein is preferentially expressed (eitherconstitutively or at a particular stage of tissue differentiation ordevelopment or in a disease state). Where the protein binds orpotentially binds to another protein or ligand (such as, for example, ina enzyme-effector protein interaction or enzyme-ligand interaction), theprotein can be used to identify the binding partner/ligand so as todevelop a system to identify inhibitors of the binding interaction. Anyor all of these uses are capable of being developed into reagent gradeor kit format for commercialization as commercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the proteins of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, proteins related to members of thesulfotransferase proteins are typically drug-metabolizing enzymes andserve as targets for identifying agents for use in mammalian therapeuticapplications, e.g. a human drug, particularly in modulating a biologicalor pathological response in a cell or tissue that expresses the proteinof the present invention. Experimental data provided in FIG. 1 showsthat the proteins of the present invention are expressed at least in thekidney and small intestine. Specifically, multiple cDNA libraries werescreened and positive clones were retrieved from libraries generatedfrom mRNA isolated from these tissues. A large percentage ofpharmaceutical agents are being developed that modulate the activity ofproteins, particularly members of the sulfotransferase subfamily (seeBackground of the Invention) and proteins that are expressed in thekideny and intestine (killing cancer calls of these tissues that expressthese proteins). The structural and functional information provided inthe Background and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data provided inFIG. 1 shows that the sulfotransferase of the present invention isexpressed at least in the kidney and small intestine. Further, theproteins of the present invention can be used in an industrial settingto label agents with tagged sulfur. Such uses can readily be determinedusing the information provided herein, that which is known in the art,and routine experimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to proteins that are related to members ofthe sulfotransferase subfamily. Such assays involve any of the knowndrug-metabolizing enzyme functions or activities or properties usefulfor diagnosis and treatment of drug-metabolizing enzyme-relatedconditions that are specific for the subfamily of drug-metabolizingenzymes that the one of the present invention belongs to, particularlyin cells and tissues that express the drug-metabolizing enzyme.Experimental data provided in FIG. 1 shows that the sulfotransferase ofthe present invention is expressed at least in the kidney and smallintestine. Specifically, multiple cDNA libraries were screened andpositive clones were retrieved from libraries generated from mRNAisolated from these tissues.

The proteins of the present invention are also useful in labeling targetagents with tagged sulfur. Such uses are readily appearent based on theenzymatic properties of the proteins of the present invention.

The proteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the protein of the presentinvention, as a biopsy or expanded in cell culture. Experimental dataprovided in FIG. 1 shows that the sulfotransferase of the presentinvention is expressed at least in the kidney and small intestine. In analternate embodiment, cell-based assays involve recombinant host cellsexpressing the protein of the present invention.

The polypeptides can be used to identify compounds that modulate theactivity of the protein in its natural state or an altered form thatcauses a specific disease or pathology associated with the protein. Boththe protein of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the protein of the presentinvention. These compounds can be further screened against a functionalprotein of the present invention to determine the effect of the compoundon the protein's activity. Further, these compounds can be tested inanimal or invertebrate systems to determine activity/effectiveness.Compounds can be identified that activate (agonist) or inactivate(antagonist) the protein of the present invention to a desired degree.Further, antagonists can be used to quench chemical reactions of theprotein (e.g. a labeling step).

Further, the proteins of the present invention can be used to screen acompound for the ability to stimulate or inhibit interaction between theprotein and a molecule that normally interacts with the protein. Suchassays typically include the steps of combining the protein of thepresent invention with a candidate compound under conditions that allowthe protein of the present invention, or fragment, to interact with thetarget molecule, and to detect the formation of a complex between theprotein and the target or to detect the biochemical consequence of theinteraction with the protein of the present invention and the target.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab)₂, Fab expression library fragments, andepitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the protein thatcompetes for substrate binding. Other candidate compounds include mutantproteins or appropriate fragments containing mutations that affectprotein function and thus compete for substrate. Accordingly, a fragmentthat competes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

Any of the biological or biochemical functions mediated by the proteinof the present invention can be used as an endpoint assay. These includeall of the biochemical or biochemical/biological events describedherein, in the references cited herein, incorporated by reference forthese endpoint assay targets, and other functions known to those ofordinary skill in the art or that can be readily identified using theinformation provided in the Figures, particularly FIG. 2. Specifically,a biological function of a cell or tissues that expresses the protein ofthe present invention can be assayed. Experimental data provided in FIG.1 shows that the sulfotransferase of the present invention is expressedat least in the kidney and small intestine. Specifically, multiple cDNAlibraries were screened and positive clones were retrieved fromlibraries generated from mRNA isolated from these tissues.

Binding and/or activating compounds can also be screened by usingchimeric protein of the present invention in which the amino terminalextracellular domain, or parts thereof, the entire transmembrane domainor subregions, such as any of the seven transmembrane segments or any ofthe intracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native protein of the present invention. Accordingly,a different set of signal transduction components is available as anend-point assay for activation. This allows for assays to be performedin other than the specific host cell from which the protein of thepresent invention is derived.

The proteins of the present invention are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the protein of the present invention (e.g. binding partners and/orligands). Thus, a compound is exposed to a protein of the presentinvention under conditions that allow the compound to bind or tootherwise interact with the polypeptide. Soluble protein of the presentinvention polypeptide is also added to the mixture. If the test compoundinteracts with the soluble protein of the present invention polypeptide,it decreases the amount of complex formed or activity from the proteinof the present invention target. This type of assay is particularlyuseful in cases in which compounds are sought that interact withspecific regions of the protein of the present invention. Thus, thesoluble polypeptide that competes with the target protein of the presentinvention region is designed to contain peptide sequences correspondingto the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the protein of the present invention, or fragment, orits target molecule to facilitate separation of complexes fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofprotein of the present invention-binding protein found in the beadfraction quantitated from the gel using standard electrophoretictechniques. For example, either the polypeptide or its target moleculecan be immobilized utilizing conjugation of biotin and streptavidinusing techniques well known in the art. Alternatively, antibodiesreactive with the protein but which do not interfere with binding of theprotein to its target molecule can be derivatized to the wells of theplate, and the protein trapped in the wells by antibody conjugation.Preparations of a protein of the present invention-binding protein and acandidate compound are incubated in the protein of the presentinvention-presenting wells and the amount of complex trapped in the wellcan be quantitated. Methods for detecting such complexes, in addition tothose described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the proteinof the present invention target molecule, or which are reactive withprotein of the present invention and compete with the target molecule,as well as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the target molecule.

Agents that modulate one of the proteins of the present invention can beidentified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

Modulators of protein of the present invention activity identifiedaccording to these drug screening assays can be used to treat a subjectwith a disorder mediated by the protein of the present inventionpathway, by treating cells or tissues that express the protein of thepresent invention. Experimental data provided in FIG. 1 shows that thesulfotransferase of the present invention is expressed at least in thekidney and small intestine. These methods of treatment include the stepsof administering a modulator of protein of the present inventionactivity in a pharmaceutical composition to a subject in need of suchtreatment, the modulator being identified as described herein.

In yet another aspect of the invention, the protein of the presentinventions can be used as “bait proteins” in a two-hybrid assay orthree-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.(1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchiet al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identifyother proteins, which bind to or interact with the protein of thepresent invention and are involved in the protein's enzymatic activity.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a protein of thepresent invention is fused to a gene encoding the DNA binding domain ofa known transcription factor (e.g., GAL-4). In the other construct, aDNA sequence, from a library of DNA sequences, that encodes anunidentified protein (“prey” or “sample”) is fused to a gene that codesfor the activation domain of the known transcription factor. If the“bait” and the “prey” proteins are able to interact, in vivo, forming aprotein of the present invention-dependent complex, the DNA-binding andactivation domains of the transcription factor are brought into closeproximity. This proximity allows transcription of a reporter gene (e.g.,LacZ) which is operably linked to a transcriptional regulatory siteresponsive to the transcription factor. Expression of the reporter genecan be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genewhich encodes the protein which interacts with the protein of thepresent invention.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a protein of the present invention-modulatingagent, an antisense protein of the present invention nucleic acidmolecule, a protein of the present invention-specific antibody, or aprotein of the present invention-binding partner) can be used in ananimal or other model to determine the efficacy, toxicity, or sideeffects of treatment with such an agent. Alternatively, an agentidentified as described herein can be used in an animal or other modelto determine the mechanism of action of such an agent. Furthermore, thisinvention pertains to uses of novel agents identified by theabove-described screening assays for treatments as described herein.

The proteins of the present invention are also useful to provide atarget for diagnosing a disease or predisposition to disease mediated bythe peptide. Accordingly, the invention provides methods for detectingthe presence, or levels of, the protein (or encoding mRNA) in a cell,tissue, or organism. Experimental data provided in FIG. 1 shows that thesulfotransferase of the present invention is expressed at least in thekidney and small intestine. The method involves contacting a biologicalsample with a compound capable of interacting with the protein of thepresent invention such that the interaction can be detected. Such anassay can be provided in a single detection format or a multi-detectionformat such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The proteins of the present invention also provide targets fordiagnosing active protein activity, disease or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered protein of the present inventionactivity in cell-based or cell-free assay, alteration in substrate orantibody-binding pattern, altered isoelectric point, direct amino acidsequencing, and any other of the known assay techniques useful fordetecting mutations in a protein. Such an assay can be provided in asingle detection format or a multi-detection format such as an antibodychip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the protein of the present inventionin which one or more of the protein of the present invention functionsin one population is different from those in another population. Thepeptides thus allow a target to ascertain a genetic predisposition thatcan affect treatment modality. Thus, in a ligand-based treatment,polymorphism may give rise to amino terminal extracellular domainsand/or other substrate-binding regions that are more or less active insubstrate binding, and protein of the present invention activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data provided in FIG. 1 shows that the sulfotransferase ofthe present invention is expressed at least in the kidney and smallintestine. Accordingly, methods for treatment include the use of theprotein of the present invention or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe proteins of the present invention, a protein comprising a fragmentof such a protein, as well as variants and fragments thereof. As usedherein, an antibody selectively binds a target protein or peptide whenit binds the target peptide and does not significantly bind to unrelatedpepetides or proteins. An antibody is still considered to selectivelybind a peptide even if it also binds to other proteins that are notsubstantially homologous with the target peptide so long as suchproteins share homology with a fragment or domain of the peptide targetof the antibody. In this case, it would be understood that antibodybinding to the peptide is still selective despite some degree ofcross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments ofthe protein of the present invention. Antibodies can be prepared fromany region of the peptide as described herein. However, preferredregions will include those involved in function/activity and/orprotein/binding partner interaction. FIG. 2 can be used: to identifyparticularly important regions while sequence alignment can be used toidentify conserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵, ¹³¹I, ³⁵S or³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data provided in FIG. 1 shows that thesulfotransferase of the present invention is expressed at least in thekidney and small intestine. Specifically, multiple cDNA libraries werescreened and positive clones were retrieved from libraries generatedfrom mRNA isolated from these tissues. Further, such antibodies can beused to detect protein in situ, in vitro, or in a cell lysate orsupernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full-length protein can be used to identify prtoein turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data provided in FIG. 1 shows that thesulfotransferase of the present invention is expressed at least in thekidney and small intestine. If a disorder is characterized by a specificmutation in the protein, antibodies specific for this mutant protein canbe used to assay for the presence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data provided in FIG. 1 shows that the sulfotransferase ofthe present invention is expressed at least in the kidney and smallintestine. The diagnostic uses can be applied, not only in genetictesting, but also in monitoring a treatment modality. Accordingly, wheretreatment is ultimately aimed at correcting expression level or thepresence of aberrant sequence and aberrant tissue distribution ordevelopmental expression, antibodies directed against the protein orrelevant fragments can be used to monitor therapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental dataprovided in FIG. 1 shows that the sulfotransferase of the presentinvention is expressed at least in the kidney and small intestine. Thus,where a specific protein has been correlated with expression in aspecific tissue, antibodies that are specific for this protein can beused to identify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the protein to a binding partner suchas a substrate. These uses can also be applied in a therapeutic contextin which treatment involves inhibiting the protein's function. Anantibody can be used, for example, to block binding, thus modulating(agonizing or antagonizing) the protein's activity. Antibodies can beprepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornucleic acids but similar methods have been developed for antibodyarrays,

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a protein of the present invention (cDNA, transcript andgenomic sequence). Such nucleic acid molecules will consist of, consistessentially of, or comprise a nucleotide sequence that encodes one ofthe proteins of the present invention, an allelic variant thereof, or anortholog or paralog thereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NOS:1and 2, alternative transcript sequences, and SEQ ID NO:3, cDNAsequences, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NOS:4-6. A nucleic acid molecule consists of a nucleotidesequence when the nucleotide sequence is the complete nucleotidesequence of the nucleic acid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQID NOS:1 and 2, alternative transcript sequences, and SEQ ID NO:3, cDNAsequences, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NOS:4-6. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NOS:1 and2, alternative transcript sequences, and SEQ ID NO:3, cDNA sequences,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ IDNOS:4-6. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided.Because of the source of the present invention, humans genomic sequence(FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acidmolecules in the Figures will contain genomic intronic sequences, 5′ and3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the proteins of the presentinvention alone, the sequence encoding the mature peptide and additionalcoding sequences, such as a leader or secretory sequence (e.g., apre-pro or pro-protein sequence), the sequence encoding the maturepeptide, with or without the additional coding sequences, plusadditional non-coding sequences, for example introns and non-coding 5′and 3′ sequences such as transcribed but non-translated sequences thatplay a role in transcription, mRNA processing (including splicing andpolyadenylation signals), ribosome binding and stability of mRNA. Inaddition, the nucleic acid molecule may be fused to a marker sequenceencoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the proteins of the present invention as well as nucleicacid molecules that encode obvious variants of the proteins of thepresent invention that are described above. Such nucleic acid moleculesmay be naturally occurring, such as allelic variants (same locus),paralogs (different locus), and orthologs (different organism), or maybe constructed by recombinant DNA methods or by chemical synthesis. Suchnon-naturally occurring variants may be made by mutagenesis techniques,including those applied to nucleic acid molecules, cells, or organisms.Accordingly, as discussed above, the variants can contain nucleotidesubstitutions, deletions, inversions and insertions. Variation can occurin either or both the coding and non-coding regions. The variations canproduce both conservative and non-conservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in FIGS. 1 and 3. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thegenomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated by thedata presented in FIG. 3, the map position was determined to be onchromosome 2 near marker SHGC-1639 (LOD score 10.01) using radiationhybrid mapping.

FIG. 3 provides SNP information that was identified from chromosomalsequence obtained from a number of individuals as well as SNPs foundduring the cDNA cloning process. These include, a6094g, t7898c, a10018c,a10791t, a12451t, t14674c, g14860c, a20133g, a1025c, t12602c, g12829t,a1442t.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloridelsodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridation conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, mapping reagents, expressioncontrol reagents and in biological assays. The nucleic acid moleculesare useful as a hybridization probe for messenger RNA, transcript/cDNAand genomic DNA to isolate full-length cDNA and genomic clones encodingthe peptide described in FIG. 2 and to isolate cDNA and genomic clonesthat correspond to variants (alleles, orthologs, etc.) producing thesame or related peptides shown in FIG. 2. As illustrated in FIG. 3,identified SNP variations include a1008c, a6094g, t7898c, a10018c,a10791t, a12451t, t14674c, g14860c, a20133g, a1025c, t12602c, g12829t,a1442t.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Figures. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asencompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence. Such molecules areuseful in genotyping and mapping experiments, particularly if the probedregion contains one or more of the sequence variants described herein.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods. As indicated by the data presented in FIG. 3, themap position was determined to be on chromosome 2 near marker SHGC-1639(LOD score 10.01) using radiation hybrid mapping. Further, the SNPvariants provided herein can be used in mapping and linkage studies.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data provided in FIG. 1 shows that thesulfotransferase of the present invention is expressed at least in thekidney and small intestine. Specifically, multiple cDNA libraries werescreened and positive clones were retrieved from libraries generatedfrom mRNA isolated from these tissues. Accordingly, the probes can beused to detect the presence of, or to determine levels of, a specificnucleic acid molecule in cells, tissues, and in organisms. The nucleicacid whose level is determined can be DNA or RNA. Accordingly, probescorresponding to the peptides described herein can be used to assessexpression and/or gene copy number in a given cell, tissue, or organism.These uses are relevant for diagnosis of disorders involving an increaseor decrease in expression relative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a protein of the present invention, suchas by measuring a level of a protein of the present invention-encodingnucleic acid in a sample of cells from a subject e.g., mRNA or genomicDNA, or determining if a protein of the present invention gene has beenmutated. Experimental data provided in FIG. 1 shows that thesulfotransferase of the present invention is expressed at least in thekidney and small intestine. Specifically, multiple cDNA libraries werescreened and positive clones were retrieved from libraries generatedfrom mRNA isolated from these tissues.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate protein of the present invention nucleic acidexpression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe protein of the present invention gene, particularly biological andpathological processes that are mediated by the protein of the presentinvention in cells and tissues that express it. Experimental dataprovided in FIG. 1 shows that the sulfotransferase of the presentinvention is expressed at least in the kidney and small intestine. Themethod typically includes assaying the ability of the compound tomodulate the expression of the protein of the present invention nucleicacid and thus identifying a compound that can be used to treat adisorder characterized by undesired protein of the present inventionnucleic acid expression. The assays can be performed in cell-based andcell-free systems. Cell-based assays include cells naturally expressingthe protein of the present invention nucleic acid or recombinant cellsgenetically engineered to express specific nucleic acid sequences.

Thus, modulators of protein of the present invention gene expression canbe identified in a method wherein a cell is contacted with a candidatecompound and the expression of mRNA determined. The level of expressionof protein of the present invention mRNA in the presence of thecandidate compound is compared to the level of expression of protein ofthe present invention mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate protein of the present invention nucleicacid expression in cells and tissues that express the protein.Experimental data provided in FIG. 1 shows that the sulfotransferase ofthe present invention is expressed at least in the kidney and smallintestine. Specifically, multiple cDNA libraries were screened andpositive clones were retrieved from libraries generated from mRNAisolated from these tissues. Modulation includes both up-regulation(i.e. activation or agonization) or down-regulation (suppression orantagonization) or nucleic acid expression.

Alternatively, a modulator for nucleic acid expression can be a smallmolecule or drug identified using the screening assays described hereinas long as the drug or small molecule inhibits the protein of thepresent invention nucleic acid expression in the cells and tissues thatexpress the protein. Experimental data provided in FIG. 1 shows that thesulfotransferase of the present invention is expressed at least in thekidney and small intestine.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe protein of the present invention gene in clinical trials or in atreatment regimen. Thus, the gene expression pattern can serve as abarometer for the continuing effectiveness of treatment with thecompound, particularly with compounds to which a patient can developresistance. The gene expression pattern can also serve as a markerindicative of a physiological response of the affected cells to thecompound. Accordingly, such monitoring would allow either increasedadministration of the compound or the administration of alternativecompounds to which the patient has not become resistant. Similarly, ifthe level of nucleic acid expression falls below a desirable level,administration of the compound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in protein of the present invention nucleic acidexpression, and particularly in qualitative changes that lead topathology. The nucleic acid molecules can be used to detect mutations inprotein of the present invention genes and gene expression products suchas mRNA. The nucleic acid molecules can be used as hybridization probesto detect naturally occurring genetic mutations in the protein of thepresent invention gene and thereby to determine whether a subject withthe mutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the protein of the present invention geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of a protein ofthe present invention.

Individuals carrying mutations in the protein of the present inventiongene can be detected at the nucleic acid level by a variety oftechniques. FIG. 3 provides SNP information that was identified fromchromosomal sequence obtained from a number of individuals as well asSNPs found during the cDNA cloning process. These include, a6094g,t7898c, a10018c, a10791t, a12451t, t14674c, g14860c, a20133g, a1025c,t12602c, g12829t, a1442t. As indicated by the data presented in FIG. 3,the map position was determined to be on chromosome 2 near markerSHGC-1639 (LOD score 10.01) using radiation hybrid mapping. Genomic DNAcan be analyzed directly or can be amplified by using PCR prior toanalysis. RNA or cDNA can be used in the same way. In some uses,detection of the mutation involves the use of a probe/primer in apolymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR) (see, e.g., Landegran et al., Science241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), thelatter of which can be particularly useful for detecting point mutationsin the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)).This method can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

Alternatively, mutations in a protein of the present invention gene canbe directly identified, for example, by alterations in restrictionenzyme digestion patterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutantprotein of the present invention gene and a wild-type gene can bedetermined by direct DNA sequencing. A variety of automated sequencingprocedures can be utilized when performing the diagnostic assays (Naeve,C. W., (1995) Biotechniques 19:448), including sequencing by massspectrometry (see, e.g., PCT International Publication No. WO 94/16101;Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al.,Appl. Biochem. Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985));Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol.217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the protein of the presentinvention gene in an individual in order to select an appropriatecompound or dosage regimen for treatment. FIG. 3 provides SNPinformation that was identified from chromosomal sequence obtained froma number of individuals as well as SNPs found during the cDNA cloningprocess. These include, a6094g, t7898c, a10018c, a10791t, a12451t,t14674c, g14860c, a20133g, a1025c, t12602c, g12829t, a1442t.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol protein of the present invention gene expression in cells,tissues, and organisms. A DNA antisense nucleic acid molecule isdesigned to be complementary to a region of the gene involved intranscription, preventing transcription and hence production of proteinof the present invention. An antisense RNA or DNA nucleic acid moleculewould hybridize to the mRNA and thus block translation of mRNA into aprotein of the present invention.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of protein of the present inventionnucleic acid. Accordingly, these molecules can treat a disordercharacterized by abnormal or undesired protein of the present inventionnucleic acid expression. This technique involves cleavage by means ofribozymes containing nucleotide sequences complementary to one or moreregions in the mRNA that attenuate the ability of the mRNA to betranslated. Possible regions include coding regions and particularlycoding regions corresponding to the catalytic and other functionalactivities of the protein of the present invention, such as substratebinding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in gene expression. Thus,recombinant cells, which include the patient's cells that have beenengineered ex vivo and returned to the patient, are introduced into anindividual where the cells produce the desired protein of the presentinvention to treat the individual.

The invention also encompasses kits for detecting the presence of aprotein of the present invention encoding nucleic acid in a biologicalsample. Experimental data provided in FIG. 1 shows that thesulfotransferase of the present invention is expressed at least in thekidney and small intestine. Specifically, multiple cDNA libraries werescreened and positive clones were retrieved from libraries generatedfrom mRNA isolated from these tissues. For example, the kit can comprisereagents such as a labeled or labelable nucleic acid or agent capable ofdetecting protein of the present invention encoding nucleic acid in abiological sample; means for determining the amount of protein of thepresent invention encoding nucleic acid in the sample; and means forcomparing the amount of protein of the present invention encodingnucleic acid in the sample with a standard. The compound or agent can bepackaged in a suitable container. The kit can further compriseinstructions for using the kit to detect a protein of the presentinvention mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, suchas arrays or microarrays of nucleic acid molecules that are based on thesequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large numberof unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit,the RNA or DNA from a biological sample is made into hybridizationprobes. The mRNA is isolated, and cDNA is produced and used as atemplate to make antisense RNA (aRNA). The aRNA is amplified in thepresence of fluorescent nucleotides, and labeled probes are incubatedwith the microarray or detection kit so that the probe sequenceshybridize to complementary oligonucleotides of the microarray ordetection kit. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

Using such arrays, the present invention provides methods to identifythe expression of the proteins of the present invention. In detail, suchmethods comprise incubating a test sample with one or more nucleic acidmolecules and assaying for binding of the nucleic acid molecule withcomponents within the test sample. Such assays will typically involvearrays comprising many genes, at least one of which is a gene of thepresent invention and or alleles of the protein of the present inventionencoding gene of the present invention. FIG. 3 provides SNP informationthat was identified from chromosomal sequence obtained from a number ofindividuals as well as SNPs found during the cDNA cloning process. Theseinclude, a6094g, t7898c, a10018c, a10791t, a12451t, t14674c, g14860c,a20133g, a1025c, t12602c, g12829t, a1442t.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the Human genome disclosed herein. Examples of such assays can befound in Chard, T, An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells, protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the Human genome disclosed herein; and (b) one ormore other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified protein of the present invention encoding geneof the present invention can be routinely identified using the sequenceinformation disclosed herein can be readily incorporated into one of theestablished kit formats which are Well known in the art, particularlyexpression arrays.

Vectors/Host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterokinase. Typical fusion expression vectors includepGEX (Smith et al., Gene 67:3140 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234(1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88 (Schultz etal., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al, EMBO J.6:187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule-sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, appropriate secretion signalsare incorporated into the vector. The signal sequence can be endogenousto the peptides or heterologous to these peptides.

Where the peptide is not secreted into the medium, the protein can beisolated from the host cell by standard disruption procedures, includingfreeze thaw, sonication, mechanical disruption, use of lysing agents andthe like. The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing a proteinof the present invention or peptide that can be further purified toproduce desired amounts of a protein of the present invention orfragments. Thus, host cells containing expression vectors are useful forpeptide production.

Host cells are also useful for conducting cell-based assays involvingthe protein of the present invention or fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native protein of the presentinvention is useful for assaying compounds that stimulate or inhibit aprotein of the present invention function.

Host cells are also useful for identifying mutants in which thesefunctions are affected. If the mutants naturally occur and give rise toa pathology, host cells containing the mutations are useful to assaycompounds that have a desired effect on the mutant protein of thepresent invention (for example, stimulating or inhibiting function)which may not be indicated by their effect on the native protein of thepresent invention.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a protein of thepresent invention and identifying and evaluating modulators of proteinactivity. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the protein of the presentinvention encoding nucleotide sequences can be introduced as a transgeneinto the genome of a non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the protein of the present inventionto particular cells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage PI. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, protein activation, and signal transduction, may not be evidentfrom in vitro cell-free or cell-based assays. Accordingly, it is usefulto provide non-human transgenic animals to assay in vivo proteinfunction, including substrate interaction, the effect of specific mutantproteins on protein function and substrate interaction, and the effectof chimeric protein of the present invention. It is also possible toassess the effect of null mutations, that is mutations thatsubstantially or completely eliminate one or more of the proteinsfunctions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

1. An isolated peptide consisting of an amino acid sequence selectedfrom the group consisting of: (a) an amino acid sequence shown in SEQ IDNOS:4-6; (b) an amino acid sequence of an allelic variant of an aminoacid sequence shown in SEQ ID NOS:4-6, wherein said allelic variant isencoded by a nucleic acid molecule that hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule shown inSEQ ID NOS:1-3 (transcript/cDNA) or 7 (genomic); (c) an amino acidsequence of an ortholog of an amino acid sequence shown in SEQ IDNOS:4-6, wherein said ortholog is encoded by a nucleic acid moleculethat hybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS:1-3 (transcript/cDNA) or 7(genomic); and (d) a fragment of an amino acid sequence shown in SEQ IDNOS:4-6, wherein said fragment comprises at least 10 contiguous aminoacids.
 2. An isolated peptide comprising an amino acid sequence selectedfrom the group consisting of: (a) an amino acid sequence shown in SEQ IDNOS:4-6; (b) an amino acid sequence of an allelic variant of an aminoacid sequence shown in SEQ ID NOS:4-6, wherein said allelic variant isencoded by a nucleic acid molecule that hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule shown inSEQ ID NOS:1-3 (transcript/cDNA) or 7 (genomic); (c) an amino acidsequence of an ortholog of an amino acid sequence shown in SEQ IDNOS:4-6, wherein said ortholog is encoded by a nucleic acid moleculethat hybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS:1-3 (transcript/cDNA) or 7(genomic); and (d) a fragment of an amino acid sequence shown in SEQ IDNOS:4-6, wherein said fragment comprises at least 10 contiguous aminoacids.
 3. An isolated antibody that selectively binds to a peptide ofclaim
 2. 4. An isolated nucleic acid molecule consisting of a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence that encodes an amino acid sequence shown in SEQ ID NOS:4-6;(b) a nucleotide sequence that encodes of an allelic variant of an aminoacid sequence shown in SEQ ID NOS:4-6, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS:1-3 (transcript/cDNA) or 7(genomic); (c) a nucleotide sequence that encodes an ortholog of anamino acid sequence shown in SEQ ID NOS:4-6, wherein said nucleotidesequence hybridizes under stringent conditions to the opposite strand ofa nucleic acid molecule shown in SEQ ID NOS:1-3 (transcript/cDNA) or 7(genomic); (d) a nucleotide sequence that encodes a fragment of an aminoacid sequence shown in SEQ ID NOS:4-6, wherein said fragment comprisesat least 10 contiguous amino acids; and (e) a nucleotide sequence thatis the complement of a nucleotide sequence of (a)-(d).
 5. An isolatednucleic acid molecule comprising a nucleotide sequence selected from thegroup consisting of: (a) a nucleotide sequence that encodes an aminoacid sequence shown in SEQ ID NOS:4-6; (b) a nucleotide sequence thatencodes of an allelic variant of an amino acid sequence shown in SEQ IDNOS:4-6, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of nucleic acid molecule shown in SEQID NOS:1-3 (transcript/cDNA) or 7 (genomic); (c) a nucleotide sequencethat encodes an ortholog of an amino acid sequence shown in SEQ IDNOS:4-6, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule shown inSEQ ID NOS:1-3 (transcriptlcDNA) or 7 (genomic); (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NOS:4-6, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acidmolecule of claim
 5. 7. A transgenic non-human animal comprising anucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising anucleic acid molecule of claim
 5. 9. A host cell containing the vectorof claim
 8. 10. A method for producing any of the peptides of claim 1comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 11. A method for producing any of the peptides of claim 2comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 12. A method for detecting the presence of any of the peptidesof claim 2 in a sample, said method comprising contacting said samplewith a detection agent that specifically allows detection of thepresence of the peptide in the sample and then detecting the presence ofthe peptide.
 13. A method for detecting the presence of a nucleic acidmolecule of claim 5 in a sample, said method comprising contacting thesample with an oligonucleotide that hybridizes to said nucleic acidmolecule under stringent conditions and determining whether theoligonucleotide binds to said nucleic acid molecule in the sample.
 14. Amethod for identifying a modulator of a peptide of claim 2, said methodcomprising contacting said peptide with an agent and determining if saidagent has modulated the function or activity of said peptide.
 15. Themethod of claim 14, wherein said agent is administered to a host cellcomprising an expression vector that expresses said peptide.
 16. Amethod for identifying an agent that binds to any of the peptides ofclaim 2, said method comprising contacting the peptide with an agent andassaying the contacted mixture to determine whether a complex is formedwith the agent bound to the peptide.
 17. A pharmaceutical compositioncomprising an agent identified by the method of claim 16 and apharmaceutically acceptable carrier therefor.
 18. A method for treatinga disease or condition mediated by a human drug-metabolizing enzymeprotein, said method comprising administering to a patient apharmaceutically effective amount of an agent identified by the methodof claim
 16. 19. A method for identifying a modulator of the expressionof a peptide of claim 2, said method comprising contacting a cellexpressing said peptide with an agent, and determining if said agent hasmodulated the expression of said peptide.
 20. An isolated human proteinsof the present invention having an amino acid sequence that shares atleast 70% homology with an amino acid sequence shown in SEQ ID NOS:4-6.21. A peptide according to claim 20 that shares at least 90 percenthomology with an amino acid sequence shown in SEQ ID NOS:4-6.
 22. Anisolated nucleic acid molecule encoding a human proteins of the presentinvention, said nucleic acid molecule sharing at least 80 percenthomology with a nucleic acid molecule shown in SEQ ID NOS:1-3(transcript/cDNA) or 7 (genomic).
 23. A nucleic acid molecule accordingto claim 22 that shares at least 90 percent homology with a nucleic acidmolecule shown in SEQ ID NOS:1-3 (transcript/cDNA) or 7 (genomic).